Simultaneous real-time imaging of oxygen gradients and in vivo microbial community spatial organization in confined environments

Abstract Microorganisms in nature are often found in porous confined environments (soils, sediments, or plant and animal tissues), self-organized in heterogeneous communities. Such an organization, resulting from complex interactions and self-generated steep chemical gradients, controls the emergent microbial community's ecological functions. The combination of microfluidics, fluorescent bacteria, and optical sensing can constitute a powerful tool to achieve new insights into these microbial dynamics. However, such a combination has remained challenging so far due to the limited compatibility of the current sensing approaches and fluorescent reporters. Here, we present a sensing microfluidic platform that enables simultaneous, real-time visualization of microscale oxygen gradients and multi-strain microbial community organization under flow. The approach combines transparent microfluidic devices, genetically encoded fluorescent bacteria, and the latest generation of near-infrared (NIR) luminescent oxygen sensors. We demonstrate that NIR oxygen sensing allows interference-free mapping of oxygen concentrations alongside GFP and mScarlet-I-labeled bacteria. Applying this platform to a heterogeneous porous geometry, we track the co-development of oxygen gradients and spatial organization in a two-strain Pseudomonas community under flowing conditions. The two strains exhibit distinct microscale spatial patterns consistent with known shape-dependent attachment and growth dynamics, while inducing sharp oxygen gradient formation. This work establishes a broadly accessible experimental framework for quantitatively linking microbial self-organization to chemical microenvironments in real time. This platform is cost-effective, customizable, and adaptable to sense different analytes, while being compatible with a range of spectrometry techniques. Therefore, this methodology opens new avenues for investigating microscale ecological processes in soils, sediments, and other confined habitats.